PROJECT SUMMARY Bacillus anthracis, the causative agent of anthrax, is a highly pathogenic bacterium considered to be a serious threat as an agent of bioterrorism. The recent use of this bacterium as a terrorist weapon in the United States exposed the need for a more effective response to this threat. B. anthracis is used as a biological weapon primarily because it forms durable spores. These spores can enter the body through multiple routes, germinate, and grow as vegetative cells, which usually results in the death of the host within several days. Natural strains of B. anthracis are sensitive to common antibiotics, which can be used to treat anthrax. However, large-scale use of antibiotics is logistically challenging and medically irresponsible. In addition, antibiotic-resistant strains could be used in future attacks. The current vaccine for anthrax has proven problematic. Thus, new strategies are needed to respond to the anthrax threat, and these are likely to require detailed knowledge of the interactions between the mammalian immune system and the outermost layer of the B. anthracis spore-the exosporium. The exosporium serves as the primary interactive site with host defenses, acts as a barrier to antibodies and destructive enzyme, and is the source of spore antigens. Additionally, it is the target of numerous detection devices. The exosporium is comprised of a paracrystalline basal layer and an external hair-like nap. The basal layer contains roughly 20 structural proteins and enzymes, and the nap is formed by the collagen-like glycoprotein BclA, the dominant antigen on the spore surface. Recent studies suggest that exosporium proteins play an important role in spore pathogenicity. The goal of this proposal is to use genetic, biochemical, and immunological methods to expand our understanding of the exosporium proteins, focusing on their critical roles in exosporium assembly, spore viability, germination, and virulence. Specifically, we will: (1) Examine the synthesis, location, and function of exosporium proteins, focusing on those with special properties (e.g., covalently modified structural proteins and germinant-degrading enzymes). (2) Elucidate the mechanism of attachment of BclA to the basal layer, which appears to occur by a novel mechanism requiring site-specific proteolytic cleavage of BclA followed by covalent attachment to a specific basal layer protein. (3) Examine the assembly of the basal layer, focusing on protein binding partners and on enzymes that covalently cross-link and modify basal layer proteins. (4) Identify the oligosaccharide attachment sites of BclA. At least two different oligosaccharides, including a pentasaccharide containing the diagnostic sugar anthrose, are O-linked to multiple sites within the collagen-like region of BclA. Our results will enable the development of new preventive and diagnostic procedures for anthrax. Additionally, our studies will reveal generally important mechanisms of protein-protein interactions, macromolecular assembly, protein attachment to cell surfaces, glycosylation and phosphorylation of bacterial proteins, and host-pathogen interactions.